The Wnt/-catenin system is essential for skeletal development in embryogenesis and regulates bone mass in adult life. However, the mechanisms by which Wnt/-catenin signaling stimulates osteogenesis and bone formation in the post-natal skeleton remain nebulous. Recent data suggest that -catenin provides critical regulatory cues at two points during the osteoblast differentiation program; in immature but committed osteoblast precursors -catenin favors maturation into matrix secreting osteoblasts and expansion of the osteoblast precursor pool; in differentiated osteoblasts it inhibits osteoclastogenesis and perhaps terminal osteoblast differentiation. In previous studies, we demonstrated that -catenin interacts with bone morphogenetic protein-2 and 4 (BMP-2/4) in producing new bone. Indeed, we find that BMP signaling is required for full osteogenic stimulation by -catenin, whereas Tcf/Lef-dependent transcriptional activity is not. We also find that -catenin is competitively recruited to either BMP or Wnt signaling pathways, and that the intersection of BMP and -catenin signaling is, at least in part, mediated by Smad4/-catenin interactions. The central hypothesis of this project is that the pro-osteogenic action of -catenin originates from its interactions with BMP signaling, and specifically Smad4, in immature osteoblasts, resulting in competitive recruitment of -catenin to either canonical Tcf/Lef-dependent or Smad4-dependent signals. Thus, -catenin can function as stimulator of either proliferation, via Tcf/Lef transcriptional activity, or maturation of immature osteoblasts, via recruitment into Smad4-containing transcriptional complexes. To test this hypothesis we propose to, 1: determine the dependency of -catenin pro-osteogenic action on BMP signaling via Smad4; 2: analyze the role of Smad4 in modulating Wnt-dependent osteogenesis and Tcf/Lef signaling; 3 analyze the molecular interactions between Smad4 and -catenin for osteogenesis. We will use in vivo and in vitro approaches based on inducible, conditional gene ablation or activation models to study whether interference with Smad4 expression alters -catenin pro-osteogenic action and canonical Tcf/Lef-dependent activity. Considering the fundamental role of -catenin in bone cell regulation, understanding the mechanisms by which -catenin delivers either a mitogenic or a differentiation signal is essential to gain a full picture of the molecular network by which bone development and homeostasis are controlled. The proposed studies will also disclose the biologic importance of a molecular interaction that may be used as a new target for bone anabolism. PUBLIC HEALTH RELEVANCE: Therapeutic options for stimulating bone formation in subjects with bone demineralization, such as osteoporosis, are limited. This research will study an interaction between two molecules that determine how bone forming cells replicate or differentiate to produce new bone. Results will allow us to better understand how bone forming cells proliferate and function in adult animals, and will disclose new ways to stimulate bone formation that may be used in the future to discover new treatments for people with low bone mass and fractures.